A chemical catalyst enabling histone acylation with endogenous acyl-CoA

Life emerges from a network of biomolecules and chemical reactions catalyzed by enzymes. As enzyme abnormalities are often connected to various diseases, a chemical catalyst promoting physiologically important intracellular reactions in place of malfunctional endogenous enzymes would have great utility in understanding and treating diseases. However, research into such small-molecule chemical enzyme surrogates remains limited, due to difficulties in developing a reactive catalyst capable of activating inert cellular metabolites present at low concentrations. Herein, we report a small-molecule catalyst, mBnA, as a surrogate for a histone acetyltransferase. A hydroxamic acid moiety of suitable electronic characteristics at the catalytic site, paired with a thiol-thioester exchange process, enables mBnA to activate endogenous acyl-CoAs present in low concentrations and promote histone lysine acylations in living cells without the addition of exogenous acyl donors. An enzyme surrogate utilizing cellular metabolites will be a unique tool for elucidation of and synthetic intervention in the chemistry of life and disease.


S11
LieD-transfected HEK293T cells were treated with 4 (100 M) for 10 h. Histone proteins were acidextracted, and acetylation levels at the indicated histone lysine residues were quantified with LC-MS/MS analysis. a The histone lysine residues within 25 Å from N-terminus of LANA peptide (residue 5-15, indicated in pink) are indicated in cyan (PDB: 5GTC). H2BK125 is not shown in this figure but is also present within 25 Å (PDB: 1ZLA). b The acetylation levels at the lysine residues in a and H2BK125, which are proximal to the LANA binding site. c The acetylation levels of other histone lysine residues. H2BK5, 11, 12, 15, 16, 20, 23, 24, and H3K115 were not analyzed due to the difficulty in detecting the digested peptide with LC-MS/MS. The mean chemical yields of the two independent experiments are shown. The overlaid dot plots present the individual data points of each experiment. The asterisks denote "not detected". Source data are provided as a Source Data file. S12

Supplementary Fig. 10 HAT enzyme-independency of H2BK120 acetylation in cells
For knockdown of H2BK120ac writer enzymes p300 (KAT3B) and CBP (KAT3A), HEK293T cells were transfected with control or p300-and CBP-specific siRNA. After transfection of the LieD protein plasmid, the cells were treated with catalyst 4 (100 M) for 10 h. H2BK120 acetylation, H3K18 acetylation, and H3K27 acetylation in the whole-cell extract were detected with anti-H2BK120ac, anti-H3K18ac, and anti-H3K27ac antibodies, respectively, by western blot analysis. Total histone proteins were visualized with Oriole staining. Representative data from two independent experiments are shown. The reduction of acetylation levels at p300/CBP target lysine residues H3K18 and H3K27 5,6 demonstrates the success of RNAi-mediated inhibition. S13

Supplementary Fig. 11 Histone-protein selectivity of mBnA-TMP(gly1) catalyst (4)-promoted acylation in cells
LieD-transfected HEK293T cells were incubated in the presence or absence of disodium malonate (NaMa, 20 mM) and then treated with 4 (100 M) for 10 h. Malonylated proteins in the whole-cell extract were detected using pan-Kma antibody by western blot analysis. Total proteins were visualized with CBB staining.
Representative data from two independent experiments are shown. S14 Supplementary Fig. 12 ChIP-qPCR analysis of H2BK120 acetylation levels at several gene regions promoted by mBnA-TMP(gly1) catalyst (4) LieD-transfected HEK293T cells were treated with 4 (100 M) for 10 h, and were analyzed by ChIP assays using anti-H2BK120ac, anti-H2B, normal mouse IgG, and normal rabbit IgG antibodies. Immunoprecipitated DNA was assessed by real-time PCR using primers specific for indicated gene locus.
The mean chemical yields of the two independent experiments are shown. The overlaid dot plots present the individual data points of each experiment. Source data are provided as a Source Data file. S15 Supplementary Fig. 13 Comparison of mBnA-and p300-mediated histone acylations in cells.
HEK293T cells were transfected with the indicated plasmids, and then treated with or without mBnA-TMP(gly1) (4, 100 M) for 10 h. Acetylated H3K18 and H2BK120, malonylated histones, and p300 in the whole-cell extract were detected using anti-H3K18ac, anti-H2BK120ac, pan-Kma, and anti-p300 antibodies, respectively, by western blot analysis. Total histone proteins were visualized with Oriole staining. Dividing

Supplementary Methods
General NMR spectra were recorded on JEOL JNM-ECX500 (500 MHz for 1 H NMR, 126 MHz for 13 C NMR) or JEOL ECS400 (392 MHz for 1 H NMR, 99 MHz for 13 C NMR) spectrometer. Chemical shifts were reported in ppm on the  scale relative to residual CHCl3 ( = 7.24 for 1 H NMR and  = 77.0 for 13 C NMR), CHD2OD ( = 3.31 for 1 H NMR and  = 49.0 for 13 C NMR), and CHD2S(O)CD3 ( = 2.49 for 1 H NMR and 39.5 for 13 C NMR) as an internal reference, respectively. Analytical HPLC was conducted by using a JASCO HPLC system equipped with a UV-2075 spectrometer, PU-2080 pumps, a DG-2080-54 degasser, and an MX-2080-32 mixer. Preparative HPLC was conducted by using a JASCO HPLC system equipped with a UV-2075 spectrometer, PU-2086 pumps, a DG-2080-53 degasser, an MX-2080-32 mixer or Shimadzu HPLC system equipped with an SPD-20A spectrometer, and LC-6AD pumps. ESI-MS spectra were measured on Agilent Technologies 6120 (for LC/MS), and Bruker micrOTOF II spectrometer (for HRMS). Absorption spectra were measured on Shimadzu UV-1800. LC-MS/MS analyses were conducted with an AB Sciex Triple TOF 4600 equipped with an Eksigent ekspert microLC 200.

LCMS
LCMS was performed using YMC-Triart C18 (2.0 mm I.D. x 50 mm) column at 40 °C with a gradient of acetonitrile in 0.1% aqueous formic acid listed below at a flow rate of 0.2 mL/min. Method: 2% Acetonitrile for 2 min, followed by a linear gradient of 2-90% acetonitrile over 13 min. UV: 230 nm.

Analytical HPLC
Analytical HPLC was performed using YMC-Pack PROTEIN-RP (4.6 mm I.D. x 150 mm) column at 40 °C with 2% acetonitrile in 0.1% aqueous TFA for 3 min, followed by a linear gradient of 2-90% acetonitrile over S20

Preparative HPLC
Preparative HPLC was performed using YMC-Triart C18 (20 mm I.D. x 250 mm) column at 40 °C with a gradient of acetonitrile in 0.1% aqueous TFA listed below at a flow rate of 10 mL/min. Method: 20% Acetonitrile for 5 min, followed by a linear gradient of 20-100% acetonitrile over 80 min. UV:

2-(Trimethylsilyl)ethyl 4-(hydroxyamino)benzoate (S8):
To a stirred solution of S7 (500 mg, 1.87 mmol) in THF (9.35 mL), rhodium 5% on carbon (50.0 mg, 0.0109 mmol) was added and then cooled to 0 °C. To the mixture, hydrazine monohydrate (109 L, 2.44 mmol) was added, and the mixture was stirred at 0 °C for 1 h. The reaction mixture was filtered through Celite to afford crude S8 solution in ca. 30 mL THF, which was used in the next step immediately without further purification.

2-mercaptoacetamido)benzamide (S14):
To a stirred solution of S13 (23.5 mg) in DCM (461 L), triisopropylsilane (TIPS, 17.7 L, 0.0865 mmol) and TFA (115 L) were added, and the mixture was stirred at r.t. for 10 min. The reaction mixture was concentrated and dissolved in water. The water layer was washed with ether, and concentrated to afford crude S14 (13.9 mg), which was used in the next step immediately without further purification.

2-(Trimethylsilyl)ethyl 3-(hydroxyamino)benzoate (S17):
To a stirred solution of S16 (587 mg, 2.20 mmol) in THF (11.0 mL), rhodium 5% on carbon (118 mg, 0.0257 mmol) was added and then cooled to 0 °C. To the mixture, hydrazine monohydrate (256 L, 5.27 mmol) was added, and the mixture was stirred at 0 °C for 1 h. The reaction mixture was filtered through Celite to S26 afford crude S17 solution in ca. 30 mL THF, which was used in the next step immediately without further purification.

Methyl 4-(hydroxyamino)benzoate (S39):
To a stirred solution of S38 (600 mg, 3.31 mmol) in THF (16.6 mL), rhodium 5% on carbon (177 mg, 0.0387 mmol) was added and then cooled to 0 °C. To the mixture, hydrazine monohydrate (386 L, 7.95 mmol) was added, and the mixture was stirred at 0 °C for 1 h. The reaction mixture was filtered through Celite to afford crude S39 solution in ca. 45 mL THF, which was used in the next step immediately without further purification.

Methyl 4-(2-bromo-N-hydroxyacetamido)benzoate (S40):
To a stirred solution of crude S39 in ca. 45 mL THF at 0 °C, NaHCO3 (2.78 g, 33.1 mmol) was added, and the mixture was stirred for 30 min. 2-Bromoacetyl bromide (1.43 mL, 16.6 mmol) in THF (5.00 mL) was added dropwise, and the mixture was stirred at 0 °C for 1 h. To the reaction mixture, water was added, and most of the THF was removed under reduced pressure. The mixture was extracted twice with EtOAc.
(6.82 mL) was added, and the mixture was stirred at r.t. for 2.5 h. After completion of the reaction, the mixture was cooled to 0 °C, and pH was adjusted to 4 with 1 N HCl aq. The mixture was concentrated, the residue was dissolved in 20% MeOH/DCM, and then filtered off. The filtrate was concentrated to afford pure S42 (140 mg, 0.580 mmol, y. 85%) as pale yellow solid (note: a ca. 10:1 mixture of cis-trans isomers at hydroxamic acid (major isomer A and minor isomer B)).

Chemical experiments
Determination of pKa values of the catalysts An appropriate amount of NaOH (3 M) or HCl (3 M) was added to Tris Buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl) to basify or acidify the solution, respectively. To the pH-adjusted solution, S1 or S2 (final 500 M) was added, the pH value was measured, and the solution was analyzed with UV-Vis spectrophotometer.

Biochemical experiments
In-cell acetylation of histone with p300/CBP siRNA treatment Eppendorf tube, centrifuged (304 g, 5 min, 4 °C), and washed with PBS. For immunoblotting of whole-cell extracts, the cells were lysed with CRB+++++ buffer on ice for 30 min, and then treated as described in "In-cell acetylation of histone with p300/CBP siRNA treatment".

S44
ChIP-assay HEK293T cells were treated as described in "In-cell acetylation of histone using endogenous Ac-CoA".
After acetylation reaction, the medium was replaced with DMEM+++. To fix the cells, 27 L of 37% formaldehyde was added per 1 mL medium. After incubation at r.t. for 15 min, 80 L of 1.25 M glycine in PBS was added per 1 mL medium to quench the fixation reaction, and the cells were harvested and washed with PBS twice. The cells were suspended in SDS lysis buffer (50 mM Tris-HCl (pH 8.1), 1% SDS, 10 mM EDTA, protease inhibitor cocktail), incubated on ice for 10 min, and sonicated with BioRaptor® II (BM Equipment Co). The supernatants were diluted 10-fold with ChIP dilution buffer (16.7 mM Tris-HCl  Supplementary Table 3. Profiling of the reactivity of lysine residues using STP ester probe Recombinant AR (0.35 M) or nucleosomes (0.175 M, 0.35 M as H2BK120) was treated with STP ester probe (1 mM) at r.t. in Tris buffer for 1 h. Proteins in the reaction mixture were precipitated by trichloroacetic acid (TCA, 16.6%). The protein was collected by centrifugation, air-dried, and dissolved in MQ. After DNA was digested by DNase I (Takara, 2270A) for 30 min at 37 °C, the samples were mixed with acetone (74%) and incubated overnight at −30 °C, then the proteins were collected by centrifugation, air-dried, and dissolved in MQ. To the solution, 50 mM aqueous ammonium bicarbonate (NH4HCO3 aq.) and 25% propionic anhydride solution (methanol/propionic anhydride, 3:1 (vol/vol)) were added, and pH was adjusted to 8 by adding ammonia solution. After 1 h incubation at r.t., the solvents were removed by Speed-Vac evaporator. The propionylated proteins were treated as described in "General method for protein digestion for LC-MS/MS analysis".

Supplementary Tables
Supplementary Table 1